Effector T lymphocytes function by secretion of mediators which influence other cells, and most of these mediators such as cytokines are secreted via the "constitutive" pathway, in which newly synthesized proteins are immediately released by exocytosis of small vesicles. In the "regulated" secretory pathway mediators are stored in cytoplasmic granules until activation signals their exocytosis, and in lymphocytes this pathway has been exclusively associated with cytotoxicity. In cloned CTL, the chemokine RANTES had been reported to use the latter pathway and to be stored in cytotoxic granules. We examined activation-induced RANTES secretion from purified subpopulations of CD8+ human T cells from blood as well as short term cultured blasts, measuring supernatant RANTES by ELISA assay. CD8+ memory and effector blood cells as well as blasts secreted RANTES within 30 minutes, while other mediators such as interferon-g required hours to be detectable. The regulated pathway was implicated for RANTES secretion by the lack of inhibition by cycloheximide and Brefeldin A. At times greater than 3 hours after TcR triggering these drugs block secretion, indicating a switch to the constitutive pathway. Flow cytometry of permeabilized cells shows that RANTES is present in cytoplasmic vesicles in the memory and effector subpopulations of CD8+ blood T cells as well as blasts, but is undetectable in the nave subpopulation. By confocal microscopy intracellular RANTES appears as vesicles that do not colocalize with proteins such as perforin or granzymes, nor with endosomal or ER markers. This lack of colocalization was confirmed by the higher resolution technique of deconvolution microscopy, both visually and by statistical analysis of digital pixel intensities. Immunogold staining of ultrathin EM sections confirmed that RANTES is present in vesicles that are morphologically distinct from the granules. When stimulated by TcR crosslinking, the RANTES positive vesicles were depleted with a half-time of 20 minutes, while at this time less than 10% of the granzyme-positive granules were depleted. Thus we have described a novel regulated secretory pathway in CD8+ T lymphocytes, capable of rapid delivery of preformed RANTES after antigen recognition. We are currently assessing the role of the regulated secretory in CD4+ T cells.